Megalin is also known as the glycoprotein 330 (gp330) or low-density lipoprotein (LDL)-receptor related protein 2 (LRP2). It is a glycoprotein expressed in the renal proximal tubular epithelial cells with a molecular weight of approximately 600 kDa (Non-Patent Documents 1 and 2).
Megalin serves as an endocytic receptor associated with endocytosis/resorption of a protein or the like in the proximal tubular lumen in the kidney before urinary excretion. A ligand of a resorbed protein or the like is then degraded by a lysosome in the proximal tubular epithelial cells (Non-Patent Document 3).
In recent years, Flavia, F. J. et al. reported, as results attained by a cell culture experiment using renal proximal tubular epithelial cells, that two types of megalins exist (i.e., full-length membrane-bound megalin and soluble megalin lacking an endodomain (a fragment containing an ectodomain)) (Non-Patent Document 4).
In addition, it has been reported that such soluble megalin (i.e., a fragment containing an ectodomain but lacking an endodomain) is constituted by a notch-like cleavage mechanism, which is mediated by the first-phase cleavage of an ectodomain in the vicinity of a cell membrane with metalloprotease and the subsequent second-phase cleavage of a transmembrane domain located inside the membrane with γ-serectase (Non-Patent Documents 5, 6, 7, and 8).
Many cell transmembrane glycoproteins have heretofore been known in which ectodomains are released from the cells to the periphery (i.e., shedding). It has been found in recent years that shedding takes place upon cleavage of a membrane protein by a given protease triggered by intracellular and extracellular signals. In addition, it has been found that such cleavage takes place in a cell transmembrane domain or endodomain in a chained manner and the cleaved fragment also functions as a signal molecule inside and outside the cell. The same applies to the megalin cleavage mechanism, and such cleavage mechanism plays a role in regulation of proximal tubular epithelial cell functions starting from the formation of a signal functional molecule (Non-Patent Documents 6 and 7). Two types of enzymes play a role in such cleavage procedures, and they are collectively referred to as metalloprotease and serectase. It has been found that activity thereof is associated with regulation of functions of various membrane proteins and is very important for diseases such as cancer or Alzheimer's disease, as well as for basic physiological functions such as cell differentiation and growth. Examples of membrane proteins in which ectodomain-shedding has been observed include tyrosine kinase growth factor receptors represented by the EGF receptor (EGFR) family, various interleukin (IL) receptors, adhesion molecules, and LDL receptors (LRP). As described above, megalin is a transmembrane glycoprotein of the LDL receptor superfamily and has been known to experience ectodomain-shedding as reported in Non-Patent Documents 5, 6, 7, and 8.
In addition, a method for measuring the amount of megalin excreted into the urine had been reported (Patent Document 1). However, patterns of human megalin, which had been subjected to cleavage modification, such as shedding by a protease in vivo, excreted into the urine have not yet been evaluated.